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(Circulation. 2000;101:2833.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Cardiovascular Responses to the Isoprostanes iPF₂-III and iPE₂-III Are Mediated via the Thromboxane A₂ Receptor In Vivo

Laurent P. Audoly, PhD; Bianca Rocca, MD; Jean-Etienne Fabre, PhD; Beverly H. Koller, PhD; Dennis Thomas, PhD; Alex L. Loeb, MD; Thomas M. Coffman, MD; Garret A. FitzGerald, MD

From Duke University and Durham Veterans Affairs Medical Centers (L.P.A., D.T., T.M.C.), Durham, NC; the Center for Experimental Therapeutics, University of Pennsylvania (B.R., A.L.L., G.A.F.), Philadelphia, Pa; and the Department of Medicine, University of North Carolina (J.-E.F., B.H.K.), Chapel Hill, NC.

Correspondence to Garret A. FitzGerald, MD, University of Pennsylvania Medical Center, Center for Experimental Therapeutics, 832 BRB II/III, Philadelphia, PA 19104. E-mail garret{at}spirit.gcrc.upenn.edu

	Abstract

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Background—Isoprostanes (iPs) are free radical–catalyzed products of arachidonic acid that reflect lipid peroxidation in vivo. Several iPs exert biological effects in vitro and may contribute to the functional consequences of oxidant stress. For example, iPF₂-III (8-iso PGF₂) and iPE₂-III modulate platelet function and vascular tone. Although these effects are blocked by antagonists of the receptor (TP) for the cyclooxygenase product thromboxane A₂, it has been speculated that the iPs may activate a receptor related to, but distinct from, the TP.

Methods and Results—Transgenic mice (TPOEs) were generated in which the TP-ß isoform was under the control of the preproendothelin promoter. They overexpressed TP-ß in the vasculature but not in platelets and exhibited an exaggerated pressor response to infused iPF₂-III compared with wild-type mice. This was blocked by TP antagonism. The platelet response to the iP was unaltered in TPOEs compared with wild-type mice. By contrast, both the pressor response to iPF₂-III and its effects on platelet function were abolished in mice lacking the TP gene. This was also true of the effects of infused iPE₂-III on mean arterial pressure and platelet aggregation.

Conclusions—Both iPF₂-III and iPE₂-III exert their effects on platelet function and vascular tone in vivo by acting as incidental ligands at membrane TPs rather than via a distinct iP receptor. Activation of TPs by iPs may be of importance in syndromes in which cyclooxygenase activation and oxidant stress coincide, such as in atherosclerosis and reperfusion after tissue ischemia.

Key Words: isoprostane • receptors • thromboxane

	Introduction

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Isoprostanes (iPs) are free radical–catalyzed prostaglandin (PG) isomers formed in situ in the phospholipid domain of cell membranes. They are cleaved by phospholipases, circulate, and are excreted in urine.^{1 2 3} Circulating iP levels are increased in reperfusion after tissue ischemia and in several disease states involving oxidative stress, including atherosclerosis, chronic pulmonary disease, Alzheimer’s disease, and diabetes mellitus.^{2 4} Tissue and plasma iPs are also increased in animals that have been exposed to carbon tetrachloride or that have been rendered deficient in antioxidants.⁵ Thus, generation of iPs reflects lipid peroxidation in vivo, and iPs have gained favor as biomarkers of oxidative stress.

In addition to their potential usefulness as indexes of oxidant stress in vivo, iPs exert effects on cells in vitro. Similar to conventional PGs that regulate cellular function via activation of distinct G protein–coupled membrane receptors,⁶ iPs may also ligate such receptors in a specific and saturable manner. Thus, 8,12-iso-iPF₂-III activates the receptor for PGF₂ (the FP), causing hypertrophy in cardiac myocytes.^{7 8} By contrast, iPF₂-III (formerly known² as 8-iso-PGF₂) activates the receptor for thromboxane A₂ (the TP⁹ ). This latter compound and the analogous iPE₂-III modulate platelet function and adhesive interactions between platelets and endothelial cells,^{10 11 12} as well as being potent vasoconstrictors in vitro and in vivo.^{13 14} All of these effects are blocked by pharmacological antagonists of the TP.

Although iPs may act as incidental ligands at membrane and nuclear¹⁵ receptors for PGs, the suggestion that they may preferentially activate specific iP receptors in vivo has attracted attention. Thus, differences in the respective potencies of iPF₂-III and structural analogs of thromboxane and PG endoperoxides, the cognate ligands of the TP, for evocation of signaling responses or functional effects mediated via the TP have been described.^{16 17} However, molecular evidence for the existence of a distinct receptor for this or any other iP has yet to be provided.

To address the hypothesis that iPF₂-III exerts its effects on platelets and vascular tone via the TP in vivo, we examined its effects in 2 mouse models in which TP expression is altered. We used transgenic mice in which vascular overexpression of the TP-ß isoform¹⁸ is directed by the preproendothelin promoter (TPOEs¹⁹ ) and mice with targeted disruption of the TP receptor gene (TP^-/-20). Whereas the pressor response to iPF₂ is enhanced in the TPOE, it is absent in the TP^-/- mouse, as are the effects of the iP on platelet function. Indeed, iPE₂-III, which has physiological effects that are similar to iPF₂-III,^{12 14} also does not cause vasoconstriction or alterations in platelet function in TP-deficient mice. These results suggest that the major cardiovascular effects of iPF₂-III and iPE₂-III are both mediated via the TP rather than by distinct iP receptors.

	Methods

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Mice
Transgenic mice overexpressing the TP selectively in the vasculature (TPOE) under the control of the preproendothelin promoter were generated by standard pronuclear injection techniques. Three independent founder lines of the TPOE were established over several generations on a B6SJLF/J strain (The Jackson Laboratory, Bar Harbor, Me). Homozygous TPOEs were identified by Southern blot analyses of genomic DNA isolated from tail biopsy samples and were confirmed by test crosses with wild-type (WT) animals. Transgene expression in the 3 founder lines was assessed by Western blot analyses of protein extracted from different organs and confirmed by ligand binding assays as described previously.²¹ The presence of the TP was detected in all 3 founder lines in the aorta, kidney, and heart, but no expression was detectable in the lungs, liver, uterus, or testis.²²

TP-deficient mice were generated by homologous recombination in embryonic stem cells as described previously.²⁰ The targeted Tp allele was detected in the offspring of these crosses by Southern blot analysis of genomic DNA isolated from tail biopsy samples. The F1 progeny carrying the mutant allele were intercrossed to obtain animals that were homozygous for the targeted mutation (TP^-/-). The TP-deficient mice used in these studies were produced by intercrossing F₂ generation animals. WT littermates were used as controls.

Isoprostane Effects on Systemic Blood Pressure
To determine the effects of overexpression of vascular TP receptors on hemodynamic responses to iPs, we compared the effects of intravenous iP infusion in TPOEs and WT mice. Mice were anesthetized with pentobarbital (Nembutal, Abbott Laboratories). After tracheotomy, a short length of polyethylene tubing was placed in the trachea to facilitate spontaneous ventilation. The carotid artery was isolated from the surrounding tissues and cannulated by use of a flexible plastic catheter. The catheter was tied in place and connected to a pressure transducer. Mean systemic arterial pressure (MAP) values were recorded continuously for the duration of each experiment. Heart rates were calculated from the arterial pressure wave as beats per minute.²³ Another catheter was inserted into the jugular vein for drug or vehicle infusions. Experiments were started 20 to 30 minutes after completion of surgical procedures. Mice received bolus injections of vehicle (0.9% sodium chloride solution), U-46619 (Cayman Chemical Co) at 10, 20, or 50 µg/kg, or the TP antagonist SQ29,548 (Cayman Chemical) at 2.5 mg/kg followed by iPF₂-III. Vehicle or drugs were injected as a 1-mL/kg solution in saline. The variability of measurements performed on the same animals after adaptation was always <10%.

In the experiments with TP^-/- mice, animals were anesthetized with isoflurane (0.8% to 1.3% vol/vol), and vascular catheters were placed as described above. After a baseline MAP determination, vehicle was injected through the venous catheter, and MAP was monitored for 5 minutes. After the vehicle injection, various doses of iPF₂-III or iPE₂-III (both obtained from Cayman Chemical and confirmed as >99% pure) were injected as a bolus.

Platelet Aggregation Studies
Mice were anesthetized with pentobarbital, and blood was obtained by cardiac puncture. Samples were centrifuged to obtain platelet-rich plasma. The supernatant was carefully removed and centrifuged again at 100g. The white interfaces (platelet-rich plasma) from both centrifugation steps were combined, and platelet counts were determined with a Coulter counter. The platelet-poor plasma supernatant was used to adjust volumes for aggregation assays. Aggregation studies were performed under constant stirring at 37°C, and light transmittance was measured with a dual-channel aggregometer.

Statistical Analyses
Data are expressed as mean±SEM. Comparisons among groups were made by ANOVA. Because initial values between the mice were expected to be slightly different, the data were normalized and expressed as a change from basal values. Differences were judged significant if P<0.05.

	Results

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Pressor Response to iPF₂-III
Basal values of MAP did not differ between TPOE and WT mice, as described previously.²² Intravenous injection of iPF₂-III (Figure 1) caused a rapid (within 20 to 30 seconds) increase in MAP in WT mice that was sustained for several minutes. As shown in Figure 2, the effects of iPF₂-III on MAP in WT mice increased in a dose-dependent manner from 10 to 50 µg/kg of the injected iP.²² In TPOE mice, this response was exaggerated. The peak increase in MAP with 50 µg/kg iPF₂-III was significantly higher in TPOE (52±20%) than in WT mice (36±8%; P<0.05) with either iPF₂-III (Figure 2) or U-46619 (167±5% versus 100±15%) as the agonist. Pretreatment of the TPOE and the WT mice with the TP antagonist SQ29,548 (2.5 mg/kg) abolished the pressor response to both iPF₂-III and U-46619 (Figure 2). The thromboxane mimetic U-46619 caused a greater pressor response than iPF₂-III in both WT and TPOEs. For example, at 50 µg/kg, U-46619 and iPF₂-III raised MAP in WT mice by 100±15% and 36±8%, respectively, compared with pretreatment levels. The comparable figures for the 2 agonists in TPOEs were 167±5% and 52±10%, respectively.

View larger version (8K): [in this window] [in a new window]   Figure 1. Continuous recordings of mean systolic arterial pressure (y axis) measured by carotid artery catheterization in representative anesthetized WT (A) and representative TPOE (B) mouse before and after bolus infusion of 50 µg/kg iPF2-III (indicated by arrow).

View larger version (20K): [in this window] [in a new window]   Figure 2. MAP (MSAP) responses of anesthetized WT () and TPOE () mice (n=3 for both groups at each concentration) to different doses of iPF2-III were measured by carotid artery catheterization. TPOE mice responded to iPF2-III with greater increases in MAP than were observed in WT. Pressor responses were prevented in WT () and TPOE () mice by pretreatment with TP antagonist SQ-29,398 (2.5 mg/kg IV), indicated by arrow. *P<0.05. All values are mean±SEM.

The effects of 50 µg/kg iPF₂-III on MAP in TP^-/- mice (n=8) and their WT controls (n=8) is shown in Figure 3A. Once again, infusion of iPF₂-III produced a brisk and significant increase in blood pressure in WT mice. By contrast, bolus injection of iPF₂-III in doses up to 50 µg/kg had no significant effect on blood pressure in TP^-/- mice.

View larger version (22K): [in this window] [in a new window]   Figure 3. MAP responses of anesthetized TP-/- and WT (both n=8) mice to iPF2-III (50 µg/kg) (A) and to iPE2-III (10 µg/kg) (B). Data are depicted as mean±SEM. C, Representative pressor responses to angiotensin II (10 µg/kg) in WT and TP-/- mice. Mean data for both groups of 8 mice depicted are shown in inset. MAP is expressed as the incremental change from a preinjection baseline.

Pressor Response to iPE₂-III
We next examined the effects of acute administration of iPE₂-III on MAP in WT and TP^-/- mice. In WTs, iPE₂ caused a dose-dependent increase in MAP. However, the pattern of the blood pressure response in WT differed from that evoked by iPF₂-III in that it was more sustained. In addition, IPE₂-III was a more potent vascular agonist. As shown in Figure 3B, 10 µg/kg iPE₂-III caused an abrupt increase in MAP in WT mice (n=8) that reached a maximum value of 16±2 mm Hg. This peak increase in pressure was greater than that caused by 50 µg/kg iPF₂-III. In the TP^-/- animals (n=8), 10 µg/kg iPE₂-III had no discernible effect on MAP.

Pressor Responses to Angiotensin II
To address the possibility that the attenuated responses to iPs in TP^-/- mice were due to a generalized defect in vasoconstrictor responses, we compared the pressor effects of angiotensin II in groups of WT and TP^-/- mice (Figure 3C). Intravenous administration of 10 µg/kg angiotensin II caused similar peak increases in MAP in WT (35.3±3.7 mm Hg) and TP^-/- (40.0±3.7 mg Hg; n=8 in each group) mice.

Platelet Aggregation Responses to iPs
Both iPF₂-III and iPE₂-III induced aggregation in platelets isolated from WT mice (Figures 4 and 5). As with the pressor response, iPE₂-III was the more potent agonist. Whereas iPF₂-III evoked a weak, reversible aggregation response, the same concentration of iPE₂-III evoked irreversible aggregation (Table). Also, iPF₂-III converted the reversible aggregation induced by 10 µmol/L ADP in WT mice to irreversible aggregation, as described previously.^{10 11} TP-deficient platelets were completely unresponsive to both iPF₂-III (Figure 4) and iPE₂-III but maintained their aggregation response to ADP (Figure 5).

View larger version (143K): [in this window] [in a new window]   Figure 4. ADP (10 µmol/L) induced reversible platelet aggregation in both WT and knockout (TP-/-) mice (top). Pretreatment of platelets with iPF2-III (10 µmol/L) induces a weak, reversible aggregation response, which, when followed by ADP (10 µmol/L), becomes irreversible. Both are absent in TP-/- mice (bottom).

View larger version (124K): [in this window] [in a new window]   Figure 5. Platelet aggregation was induced by iPE2-III in WT but not in TP-/- mice. Platelets deficient in TP remain responsive to ADP.

View this table: [in this window] [in a new window]   Table 1. Platelet Aggregation Responses

	Discussion

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iPs differ from traditional PGs in a number of respects. They are formed initially in situ in the cell membrane, not after release of arachidonic acid into the cytosol, and they may contribute to the biophysical alterations in membrane function that are characteristic of oxidant injury.^{1 3} Second, they are a much more complex family. Thus, up to 64 F₂-iPs may be formed,^{2 24} and similar families of isomers of other PGs, lipoxygenase, and epoxygenase products are likely to be generated.^{2 25 26} This level of complexity constrains the interpretation of experiments conducted with single isomers, especially when one attempts to understand their likely role as autacoids in vivo. This also pertains to their potential role as signaling molecules¹⁵ or reactive intracellular intermediates.^{27 28} Nonetheless, several biological functions have been ascribed to individual iPs. Thus, iPF₂-III is a vasoconstrictor and reduces single nephron glomerular filtration rate in micropuncture studies by predominantly affecting afferent resistance.¹⁶ It also modulates platelet function.^{10 11 12} All of these effects are blocked by pharmacological inhibitors of the TP.

The TP is the product of a single gene.²⁹ However, 2 splice variants, termed and ß and differing by the length of their carboxy terminal tails,^{8 18} have been described. Little is presently known of their discriminate functions save that they couple differentially to G_h (a high-molecular-weight G protein) or transglutaminase II³⁰ and differ in their rates of agonist-induced internalization.³¹ Both can be activated by iPF₂-III but less potently than by analogs of the cognate TP ligands, PGH₂ and thromboxane A₂.^{11 16} Thus, iPF₂-III may function as an incidental ligand at the TP. Interestingly, the FP is not activated by iPF₂-III but can be ligated by other F₂-iPs, such as 8,12-iso-iPF₂-III.⁷ The functional importance of activation of the TP by iPs in vivo is unknown. However, it is of interest that TP antagonism is more effective than doses of aspirin that completely inhibit platelet thromboxane generation in accelerating the response to thrombolysis.³² This was observed in a canine model of coronary artery thrombosis,³³ in which augmented generation of iPF₂-III is known to occur during reperfusion with the thrombolytic drug.

Several lines of evidence suggest that iPF₂-III might activate a high-affinity receptor distinct from but related to the TP. For example, whereas iPF₂-III displaces a radioactively labeled TP antagonist from binding sites in rat aortic smooth muscle cells with lesser potency than does a thromboxane mimetic, it more potently stimulates DNA synthesis and inositol 1,4,5-triphosphate formation in these cells. These effects are only partially blocked by a TP antagonist.¹⁶ By contrast, a PGH₂ mimetic, but not iPF₂-III, stimulates inositol 1,4,5-triphosphate production in rat mesangial cells, a system in which binding sites for a radiolabeled TP antagonist but not for labeled iPF₂-III were identified.¹⁷ However, the labeled iP had quite low specific activity.

To investigate receptor activation by iPs in vivo, we used mouse models that either overexpressed the ß-isoform in the vasculature or that lacked both isoforms in all tissues owing to deletion of the TP gene.²⁰ Infusion of both iPs increased MAP in WT mice, although the pattern of the hypertensive response differed. Consistent with their relative potencies as in vitro vasoconstrictors,³⁴ the peak hypertensive response to 10 µg/kg iPE₂ was roughly equivalent to that induced by 50 µg/kg iPF₂-III in vivo. Several lines of evidence suggest that the pressor response to iPF₂-III is transduced via the TP. Thus, the response is increased in TPOEs compared with WTs, in which the pressor response to infusion of an -adrenoreceptor agonist, phenylephrine, is unaltered.²² This response is abolished by pretreatment with a TP antagonist. Although this observation demonstrates that the ß-isoform of the TP can transduce the effect of the iP, both isoforms are transcribed in vascular cells in culture,³⁵ and, if translated, it is possible that either or both may be activated in the vasculature of WT animals. Conclusive evidence that the effect is mediated via the TP is that the pressor response to iPF₂-III is completely abolished in TP^-/- mice, which lack both isoforms of the receptor but respond normally to other vasoconstrictors, such as angiotensin II. Interestingly, despite a more sustained hypertensive response to iPE₂-III in WTs, the response to this iP is also abolished in TP^-/- mice. Experiments in vitro indicate that an iP and a PG may activate both overlapping and distinct downstream signaling pathways linked to a PG receptor.⁶ Such differential activation of signaling pathways may result from varied agonist-induced receptor–G-protein interactions.³⁶ This might explain the different hypertensive responses to the 2 iPs mediated via the TP in vivo in the present study. It might also underlie the discordant signaling and functional effects of PG/thromboxane mimetics compared with the iPs, which has been advanced as evidence for distinct iP receptors.^{16 17}

The effects of both iPs on platelet function also appeared to be mediated via the TP. As previously described, both iPs alone induced platelet aggregation, and iPE₂- III appears to be the more potent agonist.^{10 11 12} They also support irreversible aggregation to subthreshold concentrations of conventional platelet agonists.^{11 12} These effects on platelet function recapitulate those transduced by the form of the TP bound reversibly by the antagonist GR 32191.³⁷ Both TP isoforms are transcribed in human platelets,³⁸ but only the -isoform appears to be translated.³⁹ In our experiments, the preproendothelin promoter does not direct gene expression in megakaryocytes, and as expected, the platelet response to both thromboxane mimetics and iPF₂-III was unaltered in TPOEs compared with WTs. By contrast, deletion of the TP abolished the platelet response to either iP. Thus, just like the vascular effects, both iPs appear to exert their influence on platelets solely via the TP and not via distinct iP receptors.

The clinical development of TP antagonists coincided with the emergence of aspirin as an effective platelet-inhibitory drug in large-scale trials of platelet-occlusive diseases. This dampened enthusiasm for more expensive compounds directed at the same pathway of platelet activation. Large-scale experience with such antagonists was limited to trials in patients who had undergone angioplasty.^{40 41} The antagonists were shown to reduce periprocedural mortality,⁴⁰ but much larger trials would have been needed to detect superiority over aspirin, perhaps owing to preservation of the capacity to generate other eicosanoids, such as prostacyclin. Similarly, the impact of these compounds on restenosis appeared to depend on whether a clinical or angiographic definition of this event was used.⁴¹ Since that time, evidence has emerged to support the role of prostacyclin as a homeostatic regulator of platelet function in vivo⁴² and of its potent inhibitory effects on vascular proliferation in response to injury.⁴³ Thus, TP antagonists may be preferable to even low doses of aspirin as platelet inhibitors, where preservation of prostacyclin biosynthetic capacity is desired. An example may be in combination with selective inhibitors of cyclooxygenase-2, which markedly suppress prostacyclin biosynthesis without concomitant platelet inhibition.^{44 45} In this case, TP antagonism,⁴⁶ unlike low-dose aspirin,⁴⁷ may also contribute to gastric cytoprotection. However, head-to-head comparisons have not been performed, and both TP antagonists and aspirin may influence gastric bleeding, owing to their similar effects on primary hemostasis. Our present results raise the additional possibility of novel targets for such drugs on the basis of activation of the TP by iPs. Potential examples of such indications include ischemia/reperfusion syndromes³² and atherogenesis,⁴⁸ in which iP generation is increased. Indeed, preliminary evidence of the efficacy of a TP antagonist in the latter condition has emerged recently.⁴⁹

In conclusion, both iPF₂-III and iPE₂-III are vasoconstrictors and modulators of platelet function. Both effects are transduced via activation of the TP in vivo in the mouse and do not depend on the existence of related but distinct iP receptors. Activation of the TP by iPs may broaden the potential efficacy of pharmacological antagonists of the TP.

	Acknowledgments

 
This study was supported by grants HL-61364 and HL-54500 from the National Institutes of Health. Dr FitzGerald is the Robinette Foundation Professor of Cardiovascular Medicine. Laurent P. Audoly is a recipient of a postdoctoral fellowship from the American Heart Association.

	Footnotes

 
Drs Audoly and Rocca contributed equally to this work.

Received September 30, 1999; revision received January 3, 2000; accepted January 25, 2000.

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